The objectives of this Project in this application are two-fold: first, to define the biosynthetic pathways by which cholesterol is converted into bile acids, and second to understand the roles of oxysterols in lipid metabolism. These studies will build on our findings in the previous grant period, which demonstrated the existence of three distinct pathways by which sterols are converted into bile acids. Each pathway differs in its substrate (cholesterol vs. various oxysterols) and in the involvement of distinct sterol 7ahydroxylase enzymes that add an essential hydroxyl group to carbon-7 of the starting sterol. We have isolated cDNAs and genes encoding three different sterol 7a-hydroxylases, and a gene encoding 3B-hydroxy-steroid oxidoreductase, which catalyzes the step in bile acid synthesis immediately downstream of 7ahydroxylation. Mice lacking two of the sterol 7a-hydroxylases have been characterized in the previous grant period. Using the techniques of homologous recombination and targeted gene disruption, we now will generate lines of mice that lack the remaining sterol 7a-hydroxylase, termed the CYP39A1 oxysterol 7a-hydroxylase, and mice that lack the 3B-hydroxy steroid oxidoreductase. We will study gene expression, cholesterol metabolism, and bile acid synthesis in these knockout animals and search for new bile acid synthesis enzymes whose activities are revealed in the mutant mice. Our studies in the mouse have shown that a majority of bile acids (80%) are synthesized by the cholesterol 7a-hydroxylase pathway in which cholesterol serves as the starting substrate. The expression of the cholesterol 7ahydroxylase gene (Cyp7al) is subject to negative feedback repression by bile acids and to positive feedforward induction by oxysterols. Research by others over the last three years has shown that nuclear orphan receptors mediate regulation of the Cyp7al gene. In particular, the liver X receptor (LXR) working in concert with oxysterols and the Cyp7al promoter binding factor (CPF) activate the gene, and the farnesoid X receptor (FXR) working in concert with bile acids and the short interaction heterodimeric partner (SHP) receptor mediate down regulation of the gene. We have obtained knockout mice that lack CPF and SHP and will characterize these animals with respect to the regulation of the cholesterol 7a-hydroxylase pathway and lipid metabolism. Oxysterols perform three important physiological functions. First, they regulate the expression of genes that participate in both sterol and fat metabolism through their inhibitory effect on SREBP processing. Second, they are intermediates in the transfer of sterols from the periphery to the liver. Third, as noted above, they are substrates for bile acid synthesis. We have isolated cDNAs and genes encoding the cholesterol hydroxylases that synthesize three naturally occurring oxysterols (24-hydroxycholesterol, 25- hydroxycholesterol, and 27-hydroxycholesterol) and will now use these tools to explore the roles of individual oxysterols in lipid metabolism. Knock out mice will be produced lacking each enzyme and will be studied as described above. Embryonic fibroblast cell lines will be established from the mutant mice and parameters of SREBP cleavage, transport and turnover will be defined. Finally, we will determine the consequences for cholesterol homeostasis of overexpression of one or more of the cholesterol hydroxylases in cultured Chinese hamster ovary (CHO) cell lines. These studies will complement those of Research projects 1-4, which focus on the SREBP pathway and the identification of mutant genes in humans with disorders of cholesterol homeostasis.